Assay device with captured particle reagent

ABSTRACT

The invention describes an assay device and assembly for detecting an analyte in a liquid sample. Each assay device in the assembly includes structure defining a well, a ligand-coated particle, and a flexible particle retaining structure for holding the particle in a captured position within the well.

FIELD OF THE INVENTION

The present invention relates to an assay device for use in detecting ananalyte in a liquid sample, and in particular, to a device having acaptured particle reagent for use in analyte detection.

BACKGROUND OF THE INVENTION

A variety of solid-phase assay tests for detection or quantitation ofsolution analytes have been proposed. In a typical test of this type, atest solution is added to a solid-phase reagent which is coated withligand molecules capable of binding specifically to the analyte, or withmolecules effective to compete with the analyte for binding toreporter-labeled anti-ligand molecules. After incubation under bindingconditions, the solid-phase reagent is washed to remove unbound samplematerial, and "developed" to allow detection of analyte oranalyte-competing molecules bound to the reagent.

In one general assay format, the solid-phase reagent is a card ormulti-well device having a number of surfaces which are coated, e.g., bychemical derivatization, with ligand molecules. In another generalformat, the solid-phase reagent is a bead or particle coated with ligandmolecules. The bead is usually carried in a well, with the varioussolution addition and removal steps involved in an assay procedure beingcarried out by careful solution transfer to and from the well.

One advantage of ligand-coated particles, in an solid-phase immunoassay,is that the particles can De prepared more easily in bulk and withgreater uniformity, in terms of ligand coating density, thanligand-coated wells or surfaces. Another advantage is that the surfaceconcentration of detectable analyte-related reporter molecules on aparticle is effectively higher than on a planar surface, both because ofthe curvature of the particle and, where the particle is opticallytransparent, because of the ability to detect reporter molecules onfront and back sides of the particle.

It would be desirable to provide an assay device which utilizesligand-coated particles or beads, to exploit the advantages discussedabove, and which also permits easy manufacture, bead capture, andflexibility in designing a system for determination of a selected groupof analytes.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an assay device for use indetecting an analyte in a liquid sample. The device includes wallstructure defining a well having a bottom wall and an upper opening, anda particle having surface-bound ligand molecules for use in analytedetection. The device further includes flexible retaining structureattached to said wall structure for retaining the particle in a capturedposition within the well, when the retaining means is in an undeformedcondition, and which can be deformed to admit the particle into thewell.

In one general embodiment, the flexible retaining structure includes atleast one, and preferably three, flexible finger-like projectionsdisposed between the bottom wall and the opening of the well, where atleast one of the projections has an inwardly facing detent dimensionedto hold the particle in a captured position, with the fingers in anundeformed condition. In a related embodiment, at least one of theflexible finger-like projections further includes a second detentpositioned and dimensioned to coact with the first-mentioned detent tohold the particle in a raised position above the bottom wall in thewell.

In another general embodiment, the particle is a torus with a centralopening, and the flexible retaining structure is a stem projecting fromsaid bottom wall, and having a flared distal portion which is deformableto allow receipt of the particle opening onto the stem.

Also in one preferred embodiment, the bottom wall includes a drainageportion from which liquid can be drawn from the well, with the particlecaptured in the well.

In another aspect, the invention includes a multi-well assembly fordetecting one or more analytes in a liquid sample. The assembly includesa plurality of wall structures, each defining a well having a bottomwall, an upper opening, and one or more baffles which separate the wellfrom one or more adjacent wells, respectively. Each baffle is formed ofoverlapping wall portions which block light transmission, but allowfluid flow, between such adjacent wells.

Each well in the assembly may be designed to hold a particle havingsurface-bound ligand molecules for use in analyte detection. In apreferred embodiment, each well includes flexible particle-retainingstructure, such as described above, for retaining the particle in acaptured condition.

In one configuration, the wells in the assembly are arranged in a row,with each pair of adjacent wells being separated by one of the baffles.In another configuration, the wells in the assembly are arranged about acentral port region, with each well being separated from this portregion by one of the baffles.

In still another aspect, the invention includes a multi-well assayassembly designed for analyte detection, based on chemiluminescencereactions occurring in adjacent wells of the device. The walls of thedevice separating adjacent wells are formed of polymeric materialcontaining 0.05-0.5 weight percent carbon black.

In still another aspect, the invention includes, a method of carryingout a plurality of solid-phase diagnostic assays for selected analytes.The method includes placing in a predetermined particle-dispensingrelationship (i) an assay assembly of the type described above havingmultiple wells with particle-capture structure, and (ii) a particledispensing device having a plurality of cartridges, each containingparticles coated with a selected ligand. A particle from one or more ofthe cartridges is dispensed into one or more selected wells in assembly,and each dispensed particle is forced into a captured position in theassociated well.

These steps are repeated until particles have been dispensed andcaptured in a plurality of the wells. The assembly and capturedparticles are now used for diagnostic assays in each of theparticle-containing wells.

These and other objects and features of the present invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a perspective view of a tray containing multi-wellassay assemblies constructed in accordance with the invention;

FIG. 2 shows a portion of one of the FIG. 1 assemblies, seen from thetop in FIG. 1;

FIG. 3 is an enlarged view of a single-well device in the FIG. 2assembly;

FIG. 4 is an enlarged sectional view of a single-well device, takenalong line 4--4 in FIG. 3;

FIGS. 5A and 5B illustrate entry (5A) and capture (5B) of a particle inthe device illustrated cross-sectionally in FIG. 4;

FIG. 6 is a cross-sectional view of a single-well device, like thatshown in FIG. 4, but designed for supporting a particle in a raisedposition in the well;

FIGS. 7A and 7B illustrate entry (7A) and capture (7B) of a toroidalparticle in a single-well assay device constructed according to a secondgeneral embodiment of the invention;

FIG. 8 shows a plan view of a portion of a connected-well assemblyhaving a linear configuration of wells;

FIG. 9 shows a plan view of a portion of a connected-well assemblyhaving a central-port configuration; and

FIG. 10 is a schematic view of illustrating a method for distributingselected-analyte beads to selected wells in an assay assembly of thetype described above.

DETAILED DESCRIPTION OF THE INVENTION A. Captured-Bead Assay Device

FIG. 1 shows, in perspective view, a tray 10 designed for holding aplurality of multi-well assemblies, such as assemblies 12, 14,constructed in accordance with the invention. In the embodiment shown,the tray is designed to hold eight such assemblies. The tray and loadedassemblies are designed to be used in a multi-well assay reader such asa multi-well luminometer which functions to move the tray in a mannerthat allows automated, sequential luminescence readings from each well.

FIG. 2 shows a plan view of a portion of assembly 12 seen in FIG. 1. Theassembly, which is representative, includes a linear array of assaydevices, such as devices 16, 18, constructed according to the presentinvention. The assembly is preferably formed as a single molded plasticarticle, with the individual devices in the assembly being connected attheir upper adjacent edges, such as adjacent edges 20, 22 in devices 16,18, respectively (FIGS. 2 and 3). In the embodiment shown, the assemblyincludes 12 assay devices, giving a total of 96 such devices in thefully loaded tray in FIG. 1.

FIG. 3 shows an enlarged plan view of device 16 in assembly 12. The samedevice is seen cross-sectionally in FIG. 4. The construction of device16, which is representative, will be illustrated with particularreference to these two figures. As seen, the device includes an elongatewell 24 formed by a rectangular wall structure or means 26. The upperedge of the wall structure defines an upper well opening 28. The lowerportion of the wall structure, has a reduced width and length whichdefines a lower well region 30 having a bottom wall 32. The side wallsforming the lower region are indicated at 34. The lower well regionincludes a particle-receiving section 32a, and a liquid-removal section32b. The bottom wall is sloped to allow fluid in the well region to flowfrom section 32a toward section 32b, for a purpose to be described.

With continued reference to FIGS. 3 and 4, device 16 includes threeflexible, finger-like projections, such as projections 36, 38 whichproject upwardly from the bottom wall of the well toward the well'supper opening. Each projection has an inwardly facing detent, such asdetent 40 in projection 36, formed at its free, i.e., upper end. Theprojections are also referred to herein, collectively, as flexibleretaining means. As shown, the three projections are formed integrallywith the side walls forming the lower well region, and encompass theparticle-receiving section of the well region.

According to an important feature of the invention, an assay particle orbead 42 in the device is received in a captured position in the lowerwell region by the flexible retaining means which comprise, in thepresent embodiment, the three flexible finger projections. The bead issupported in the well in a slightly elevated position on a protuberance35 formed on the bottom of the well, to increase liquid circulationabout the particle surface, and enhance analyte capture at surface-boundligand molecules on the bead in an analyte assay.

The bead is preferably a conventional glass or polymeric bead to whichbiological assay materials, specifically, surface-bound ligandmolecules, can be attached. Typically, the beads have a diameter ofbetween about 1-7 mm. Details of the preparation of the beads, and theirrole in a particle assay, are given below.

FIGS. 5A and 5B illustrate insertion and capture of bead 42 in device12. As seen, the bead dimension is such as to force the three fingerprojections outwardly, to accommodate entry of the bead past thefinger-projection detents (FIG. 5A), but allow seating of the bead atthe bottom of the well (FIG. 5B). In its captured position, shown inFIG. 5B, the bead is free to rotate and to move vertically between itsat rest position on the bottom of the well (in contact with protuberance35) and a raised position in contact with the lower surfaces of thefinger-projection detents.

The construction and scale of the device is preferably such as to allowassays to be performed with small liquid sample volumes, such as 50 to1000 microliter volumes, sufficient to fill the lower well portion ofthe device, at least to a level covering bead 42.

As indicated above, the device is preferably formed as a molded polymerarticle, and also preferably part of a multi-device assembly, such asassembly 12. Where the assembly is employed for chemiluminescent assays,the material forming the assembly is preferably an opaque polymer, suchas polyethylene or polyacrylate, containing 4-15 weight percent titaniumdioxide. In accordance with one aspect of the invention, it has beendiscovered that the wells in an assembly can be made completelyrefractory to chemiluminescent light transmission between wells byaddition of between 0.0005 and 0.5 weight percent carbon black to thepolymer material used in forming an assembly. The invention includes, inone aspect, a chemiluminescent assay device constructed of an opaquepolymer containing titanium dioxide and also containing between 0.0005to 0.5 weight percent carbon black.

FIG. 6 shows, in a cross-sectional view similar to that in FIG. 4, asecond embodiment of a captured-bead device 46 formed in accordance withthe invention. The device has the same general construction as device 12above, including an identical wall structure 47 defining a well 49. Thedevice differs from device 12 only in the construction of the threeflexible, finger-like projections, such as projections 48, 50, used forcapturing a particle or bead 52 in the device.

Projection 48, which is representative, has an upper detent 54, similarto detent 40 in projection 36, and a lower detent 56 which isdimensioned, in combination with the lower detents of the other twoprojections in the device, to capture the bead between the two sets ofdetents, as shown. The bead can be moved to a second "reaction" positionbelow the lower detents by forcing the bead over the lower detents. Thepurpose of the two-position feature in an assay will be described below.

A third embodiment of a bead-capture device constructed according to theinvention, and indicated generally at 58, is illustrated in FIGS. 7A and7B. The device includes wall structure 60 defining a well 62,substantially identical to the wall structure in device 12. The devicediffers from device 12 in the construction of the assay particle andmeans for retaining or capturing the particle in the device.

Specifically, the retaining means includes a single vertically extendingstem or post 70 which has flexibly movable flared portions 74 cut fromand extending away the upper end of the post, in a relaxed condition, asseen in FIG. 7B.

The ligand-coated particle in the device is a toroidal-shaped particle76 whose interior hole is dimensioned to slide freely over post 70. Toplace the particle in a captured condition in the device, the particleis placed on the post, as shown in FIG. 7A, with downward movementserving to press portions 74 against the side of the post, in effectdeforming the relaxed condition of the portions 74. Once the particle isreceived on a lower region of the post, the flared portions return totheir relaxed conditions, shown in FIG. 7B, acting to prevent theparticle from sliding off the post.

In operation of the devices described above, a coated particle, such asparticle 42 in FIG. 2, coated with a selected analyte-related ligand, isplaced in the well of the device, where the particle becomes captured inthe well. The series of sample and test solutions are then added to andremoved from the well, employing selected incubation times and reactiontemperatures suitable for the particular assay. One exemplary test fordetection of a selected-sequence DNA analyte is described in the examplebelow.

As noted above, the particle in the well is exposed fully to each sampleor test solution added to the well, by virtue of the slight elevation ofthe particle in the well, and the ability of the particle to rotatefreely in its captured condition. After a selected incubation time,fluid in the well is removed by aspirating from the liquid-removalsection 32b of the well, this section acting to drain liquid from theparticle-receiving section of the well, to promote substantiallycomplete liquid removal.

The retaining means in the device also acts to restrain the particle ina desired reaction position when sample or test solution is added to thewell. In the embodiment illustrated in FIG. 6, the particle may beadditional placed at a captured position in the upper portion of thewell, to allow chemical-solution mixing and reaction in the well withoutexposure to the particle.

After the cycle of assay solutions and reactions are completed, theparticle is assayed for analyte-related surface reporter groups,typically with the particle submerged in a suitable detection solution,e.g., an enzyme substrate where the captured reporter is an enzyme, asillustrated for the chemiluminescent assay described in the example.

After the assay procedure, the particle may be removed from the well,allowing the device to be reused when washed and loaded with a freshparticle.

B. Connected Well Assembly

In another aspect, the invention includes a multi-well assay assemblyhaving fluid-connected, but light-isolated wells.

FIG. 8 illustrates an assembly 80 formed according to one embodiment ofthe invention. The assembly includes a multi-well housing 82 havingintegrally formed side walls, such as walls 84, 86, end walls, such asend wall 88, and a bottom wall 90, collectively forming an elongatechannel 92. The channel is divided into a plurality, typically 4-8,wells, such as wells 94, 96 by baffles, such as baffle 98.

As shown, each baffle is formed of a pair of overlapping wallextensions, such as extensions 100, 102 forming baffle 98 and extendingfrom side walls 84, 86, respectively. As can be appreciated from thefigure, the baffles allow flow of liquid between adjacent wells, butblock direct light transmission from center regions of adjacent wells.The upper edges (those seen in FIG. 8) of the wall portions and bafflesdefining each well form an upper opening in each well, such as opening104 in well 94.

In each well, such as well 92, there is provided flexible retainingmeans for retaining a ligand-coated particle, such as particle 106 shownin dotted lines in the figure. In the embodiment shown, the retainingmeans includes three finger-like projections, such as projections 108,110, which are formed integrally with and project upwardly from thebottom wall of the assembly. Each projection has an inwardly facingdetent, such as detent 112 on projection 110, dimensioned to hold theparticle in a captured position within the well, when the projectionsare in a relaxed, i.e., undeformed conditions. The fingers can bedeformed outwardly, as described above with respect to device 16, toaccommodate entry of a particle into the well.

The assembly, including the wall structures, baffles, and retainingmeans, are preferably formed as a integrally as a molded or injectedopaque polymer article. In an assembly employed for chemiluminescentassays, the material forming the assembly preferably contains 0.05 to0.5 weight percent carbon black, as above, to prevent lightcontamination through the walls or baffles.

The wall structure defining each well, the retaining means in that well,and the particle captured within the well by the retaining means arealso referred to herein as an assay device, such as device 114. Suchdevice differs from device 16 described above in that (a) the bottomwalls of each well are flat, rather than sloped, and (b) at least onedefining side wall in the device is a baffle, such as baffle 98.

In operation, a coated particle, such as particle 106 is placed in eachwell of the assembly, where the particle becomes captured in the well.According to an important feature of the invention, the sample and testsolutions can be added to and removed from the wells in the assemblyfrom a single well, e.g., well 94, with fluid freely circulating to allof the wells during liquid addition, and being drawn from all of thecells during liquid removal. The advantage of this approach is that allof the wells receive the same solution with each fluid change, improvingthe uniformity of assay conditions among the several wells.

After the cycle of assay solutions and reactions are completed, theparticle is assayed for analyte-related surface reporter groups, asabove. Following an assay procedure, the particle may be removed fromthe well, allowing the device to be reused when loaded with a freshparticle.

FIG. 9 illustrates another embodiment of a connected-well assembly, hereindicated at 116. The assembly includes a multi-well housing 118 havingan outer wall 120 formed by four intersecting U-shaped wall portions,such as wall portion 122, and a bottom wall 124. Each wall portion, suchas wall portion 122, the underlying bottom wall, and the inwardlyprojecting end regions of adjacent wall portions, such as end regions128, 130, form a well, such as well 126, having an upper well opening,such as opening 132. The two end regions defining the inner wall of eachwell are angled, as shown, to form a baffle, such as baffle 138 in well126, which allows fluid flow, but blocks light transmission between thewell and a central region 136 of the assembly.

The shape of the well is substantially like that of above-described well24 in device 16, in that the well has a lower, reduced cross-sectionregion, and a bottom wall which slopes downwardly from aparticle-receiving region, such as region 134 in well 126, toward thecenter region of the assembly.

In each well in the assembly, such as well 126, there is providedflexible retaining means for retaining a ligand-coated particle, such asparticle 146 shown in the figure. In the embodiment shown, the retainingmeans includes three finger-like projections, such as projection 140,which are formed integrally with and project upwardly from the bottomwall of the assembly. Each projection has an inwardly facing detent,such as detent 142 on projection 140, dimensioned to hold the particlein a captured position within the well, when the projections are in arelaxed, i.e., undeformed conditions. The fingers can be deformedoutwardly, as described about with respect to device 16, to accommodateentry of a particle into the well.

The wall structure defining each well, the retaining means in that well,and the particle captured within the well by the retaining means arealso referred to herein as an assay device, such as device 148. Eachdevice is substantially as above described device 16, except for theprovision of an inner wall separated from a central area by alight-tight baffle. The assembly is preferably formed as a single moldedarticle, as above.

Operation of the assembly in a multi-sample assay procedure is asdescribed for assembly 80 above, with sample and test liquid being addedto and removed from the assembly from central region 136.

C. Coated Particles

Particles employed in the assay devices described above are preferablyspherical-bead or toroidal particles. The particles are prepared usingknown, defined size glass or polymer beads, such as polystyrene,polyacrylamide, polymethyl acrylate, derivatized cellulose fibers,carboxylated polystyrene, polyvinylchloride, polymethylacrylate,polypropylene, latex, polytetrafluorethylene, or polyacrylonitrilebeads. In a preferred embodiment, particles are polystyrene beads havingground surfaces to minimize nonspecific molecule binding to the particlesurfaces. The particles have a preferred size between about 1-7 mm,preferably about 2-5 mm.

The analyte-related ligand which is bound to the particle surface is oneselected to bind specifically with an analyte or a reagent which iscapable of competing with the analyte for binding to the particle-boundligand. The ligand is attached to the particle surface, eithercovalently or by adsorption, using conventional chemical coupling orsurface adsorption methods. One exemplary method for use in attachingDNA ligand molecules to polystyrene beads is given in the example below.

Analyte molecules to be detected typically are nucleic acids, either DNAor RNA, or antigen or antibody analytes. The nucleic acid analytes maybe derived from RNA or DNA viruses, bacteria, fungi, or the like, whichcan infect a specific host. Additionally, the analytes may be derivedfrom the genome of a host which is suspected to contain alleles,mutations or lesions which may make the host susceptible to disease.

The nucleic acid usually contains between 20 and 2000 bases, preferablybetween 30 and 500 bases. In some cases it may be necessary to digestthe sample nucleic acid to the appropriate size prior to thehybridization step. If the nucleic acid analyte is present indouble-stranded form the nucleic acid molecule is converted to itssingle-stranded form by treatment with heat and sodium hydroxide priorto hybridization.

The nucleic acid analytes will typically contain at least one region ofsequence complementarity with the nucleic acid ligand molecule which areat least 10 nucleotides in length and up to 1000 nucleotides in length,but typically are of lengths between 15 and 200 nucleotides.

Alternatively, the analytes may be antigens, or other molecules that canbe detected by use of antibodies reactive with the molecules. To bindthese analytes to a particle surface, first antibodies reactive with theanalytes are attached to the surface. To detect surface-bound analytesthe particles are incubated with a second a antibody that incorporates asignal-generating label.

In one embodiment of the invention, the particle has a selected specificgravity, e.g., 1 to 1.05, which allows the particle to be floatedbetween raised and lowered captured position, according to the densityof assay liquid reagents which are added to wells. For example, it maybe desirable to completely submerge the sample during initial phases ofan assay, but float the particle to the top of liquid in the well fordetecting reporter binding to the particle, at the final step in anassay procedure.

D. Particle-Loading Method

One advantage of the assay assembly described above is the ability toprepare the assembly, on site, for use in a variety of differentanalyte-assay formats. In one aspect, the invention includes a methodfor carrying out a plurality of solid-phase diagnostic assays forselected analytes. The method will be described with reference to FIG.10.

The figure shows a portion of a multi-well assay assembly 150 of thetype described, for example, with respect to FIG. 2. Briefly, theassembly includes a plurality of well-defining structures, such asstructures 152, 154, which define assay wells and provide flexibleparticle-retaining structures, such as structure 156. The assembly iscarried on a movable platform 158 which can be shifted in the directionof arrow 160 to place the wells in the assembly at selectedparticle-receiving positions.

Particles are dispensed into the wells by a particle dispensing device162 having a plurality of cartridges, such as cartridges 164, 166. Eachcartridge can be loaded with particles, such as particles 168 incartridge 166, that are coated with a selected ligand useful forassaying a particular selected analyte. For example, if the analyte tobe assayed is a selected antigen, the ligand may be an antibody specificagainst the analyte antigen. Preferably each cartridge suppliesparticles having a distinctive surface-bound ligand. The particles areretained in each cartridge by a flexible lip, such as lip 171, at thebottom of cartridge 116.

Each cartridge is shiftable, e.g., under solenoid or pneumatic control,between a raised position, shown for four of the plungers, and alowered, dispensing position, as shown for plungers 164, 166.

Associated with each cartridge, and shiftable therewith, is a plunger,such as plunger 168 associated with cartridge 164. Each plunger includesa piston, such as piston 170 in plunger 168, which is shiftable, e.g.,under solenoid or pneumatic control, from a raised position to aplurality of lowered positions, for dispensing particles from thecartridge, as will be described below.

In operation the assay assembly is shifted to a selected position atwhich one or more wells in the assembly is positioned to receive aselected-ligand particle from one or more cartridges. Once positioned,the selected cartridge(s) are lowered to a position shown for cartridges164, 166 in the FIG. 10. For each selected cartridge, the associatedpiston is shifted to dislodge the lowermost particle from the cartridge,thus dispensing the particle into the well (as shown for cartridge 164in FIG. 10), with continued piston movement acting to force thedispensed particle into a captured position in the well (as shown forcartridge 166 in FIG. 10).

After this particle dispensing operation, each cartridge is moved to itsraised position, and the assembly is moved, by shifting platform 158, toa new position at which one or more unfilled wells are positioned toreceive a selected ligand-coated particle from one or more of thecartridges. This process is repeated until a desired number of wells hasbeen filled with selected particles. The assembly is then used inconducting multi-analyte assays, as described above.

The following example illustrates a nucleic acid analyte assay carriedout using coated beads of the type suitable for use in the presentinvention. The example is intended to illustrate, but in no way limit,the scope of the invention.

Example Bead Assay for HIV Transcript

A. Preparation of Coated Beads

A batch of 1000 3.12 mm polystyrene beads obtained from Hoover PrecisionPlastics (Sault Ste. Marie, Me.) were soaked in 200 ml of 1 N HCl forone hour at room temperature with agitation at 100 RPM. After washingone time with 1X phosphate buffered saline, the beads were soaked in 200ml of 1 N NaOH for one hour at room temperature with agitation at 100RPM. The beads were then washed four times with 1X phosphate bufferedsaline.

Poly(phe-lys) was purchased from Sigma Chemicals, Inc. (St. Louis, Mo.).This polypeptide has a 1:1 molar ratio of phe:lys and an average m.w. of47,900 gm/mole. It has an average length of 309 amino acids and contains155 amines/mole. To 1000 beads was added 10 mg of poly(phe-lys) in 50 mMsodium phosphate buffer pH 7.8 and the mixture was rocked overnight at4° C.

To 10,000 picomoles of the following oligonucleotide: (SEQ ID NO:1)3'-CACTTCACTTTCTTTCCAAGAGX-5, (X is the long chain amine modifiednucleotide (N⁴ -(6-aminocaproyl-2-aminoethyl) derivative of5-methylcytidine)) in 50 mM sodium phosphate pH 7.8 was added 15 mgbis(sulfosuccinimidyl) suberate (BS³). The mixture was vortexed andincubated at room temperature for 30 min. A gel filtration column(NAP-25; Pharmacia) equilibrated with 10 mM sodium phosphate pH 6.5 wasused to purify the activated oligonucleotide. The activatedoligonucleotide reaction mixture was applied to the column and allowedto filter. The eluate was collected and diluted to 100 ml with 50 mlsodium phosphate, pH 7.8 and added to the beads. The mixture wasincubated at 4° C. overnight with agitation. The beads were then washedfour times with 1X phosphate buffered saline.

In order to strip the derivatized bead of loosely bound DNA, thefollowing wash steps were employed. 1000 beads were processed in a 300ml final volume of 0.2N NaOH, 0.5% SDS for 1.5 hours at 63° C. withagitation. After cooling at room temperature for 10 min the beads werewashed four times with 1X phosphate buffered saline. The beads were thenovercoupled using 50 mg/100 ml of BS³ in 50 mM sodium phosphate buffer,pH 7.8, at 4° C. overnight with agitation. The beads were washed fourtimes with 1X phosphate buffered saline, then dried at 37° C. and storedat 4° C.

B. Hybridization Assay

As a target, a synthetic RNA transcript (containing the pol region ofHIV-1 sequence) was prepared and used at 1.2×10⁶, 6.0×10⁵ and 3×10⁵copies/ml. The negative control contained no target RNA.

Sample preparation consisted of delivering 300 μl of 60 units/mlproteinase K in 175 mM HEPES, pH 7.5, 14 mM EDTA, 1.75% lithium laurylsulfate, 1.025M lithium chloride, and 33 fmoles capture extender probesspecific for HIV pol region (each), 555 fmole label extender probesspecific for HIV pol region (each) to each well containing a DNA coatedbead. 100 μl of RNA in the amounts listed above, diluted in negativehuman plasma, was then added to the wells. Wells were agitated to mixthe contents, covered and incubated for 16 hr at 63° C.

After a further 10 minute period at room temperature, the contents ofeach well were aspirated to remove all fluid, and the wells washed 2×with washing buffer (0.1% SDS, 0.015M NaCl, 0.0015M sodium citrate). Abranched DNA signal amplification multimer was then added to each well(50 μl of 600 fmol/ml solution in 0.6M NaCl, 0.06M sodium citrate, 1.3%SDS, 50% horse serum). After covering the plates and agitating to mixthe contents in the wells, the plates were incubated for 30 min at 53°C.

After a further one minute period at room temperature, the wells werewashed as described above.

Alkaline phosphatase label probe, disclosed in EP 883096976, was thenadded to each well (50 μl of 500 fmol/ml solution in 0.6M NaCl, 0.06Msodium citrate, 1.3% SDS, 50% horse serum). After incubation at 53° C.for 15 min, and 10 min at room temperature, the wells were washed threetimes as above and then three times with 0.015M NaCl/0.0015M sodiumcitrate.

An enzyme-triggered dioxetane (Schaap et al., Tet. Lett. (1987)28:1159-1162 and EPA Pub. No. 0254051), obtained from Lumigen, Inc., wasemployed. 50 μl Lumiphos 530 (Lumigen) was added to each well. The wellswere tapped lightly so that the reagent would fall to the bottom andgently swirled to distribute the reagent evenly over the bottom. Thewells were covered and incubated at 37° C. for 20-40 min.

The light output from the dioxetane reagent was then read on aluminometer. Output was given as the full integral of the light producedduring the reaction, with the results given in the table below,.

    ______________________________________                                        HIV RNA Copies/ml                                                                             Luminescence Units                                            ______________________________________                                        1.2 × 10.sup.6                                                                          1.6                                                           6.0 × 10.sup.5                                                                          0.92                                                          3.0 × 10.sup.5                                                                          0.53                                                          0               0.11                                                          ______________________________________                                    

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

It is claimed:
 1. An assay device for use in detecting an analyte in aliquid sample by means of a detectable reaction that is related to theamount of analyte present in the sample, comprisingwall means defining awell having a bottom wall and an upper opening through which such areaction can be detected, a particle having surface-bound ligandmolecules for use in analyte detection, said particle being supported onsaid bottom wall, and flexible retaining means attached to said wallmeans, for retaining said particle in a captured position within saidwell, when the retaining means is in an undeformed condition, and whichcan be deformed to admit said particle into the well.
 2. The device ofclaim 1, wherein said flexible retaining means includes finger-likeprojection means extending from said bottom wall in the direction ofsaid upper opening.
 3. The device of claim 2, wherein said finger-likeprojection means includes three flexible projections extending from saidbottom wall in the direction of said upper opening and terminating atfree ends, where at least one of said projections has an inwardly facingdetente dimensioned to hold the particle in a captured position, withsaid projections in an undeformed condition.
 4. The device of claim 3,wherein at least one of the three flexible finger-like projectionsfurther includes a second detent positioned and dimensioned to coactwith the first-mentioned detent to hold said particle in a raisedposition above said bottom wall in said well.
 5. The device of claim 1,wherein said particle is a torus with a central opening, and saidflexible retaining means is a stem projecting from said bottom wall, andhaving a flared distal portion which is compressible to allow receipt ofthe particle opening onto said stem.
 6. The device of claim 1, whereinsaid bottom wall includes a drainage portion from which liquid can bedrawn from the well, with the particle captured in the well.
 7. Thedevice of claim 1, which further includes a means formed on said bottomwall, effective to support said particle in an elevated position in thewell.
 8. The device of claim 1, for use in detecting a nucleic acidanalyte, wherein the ligand molecules are single-stranded nucleic acidmolecules including sequence complementarity to said nucleic acidanalyte.
 9. The device of claim 1, wherein said analyte is detected by achemiluminescence detector, and said wall means are constructed of anopaque polymer.